LOCUS       sequence-180151-        3044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express sgRNA targeting human CYFIP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3044)
  AUTHORS   Hazelbaker DZ, Beccard A, Bara AM, Dabkowski N, Messana A, Mazzucato
            P, Lam D, Manning D, Eggan K, Barrett LE
  TITLE     A Scaled Framework for CRISPR Editing of Human Pluripotent Stem 
            Cells to Study Psychiatric Disease.
  JOURNAL   Stem Cell Reports. 2017 Oct 10;9(4):1315-1327. doi: 
            10.1016/j.stemcr.2017.09.006.
  PUBMED    29020615
REFERENCE   2  (bases 1 to 3044)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stemcr.2017.09"; journalName: "Stem Cell Reports"; date: 
            "2017-10-10- 10"; volume: "9"; issue: "4"; pages: "1315-1327"
COMMENT     Sequence Label: pPN234
FEATURES             Location/Qualifiers
     source          1..3044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        292..367
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(392..408)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(392..408)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(405..427)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    416..432
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(440..446,447..464,465..470))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    485..506
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(623..640)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(794..1382)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(874..893)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1553..2344,2345..2413))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2176..2195
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2414..2518)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2586..2604
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2642..2664)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2764..2783
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2977..2999
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2991..3008
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2992..3008
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gctgctcgtt acatttcacc gtttgctcgt tacatttcac cgttttagag
      301 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      361 tcggtgcttt tttaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
      421 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      481 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
      541 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
      601 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      661 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      721 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      781 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      841 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      901 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      961 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1021 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1081 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1141 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1201 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     1261 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     1321 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1381 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1441 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     1501 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     1561 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     1621 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     1681 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     1741 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     1801 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     1861 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     1921 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     1981 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     2041 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     2101 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     2161 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     2221 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     2281 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     2341 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     2401 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2461 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     2521 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     2581 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     2641 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     2701 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     2761 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     2821 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
     2881 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     2941 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     3001 cggccagtga attcgagctc ggtacccggg gatccgttaa ttaa
//
